The goal of this proposal is to develop methods for studying replication and molecular genetic analysis of the minus-strand plant rbabdovirus, sonchus yellow net virus (SYNV), whose genome structure shares many common features with those of animal rhabdoviruses, such as rabies and vesicular stomatitis virus. The proposal has two objectives: Objective 1 will be to determine the subcellular distribution of viral proteins in plant cells at both the early and late stages of viral morphogenesis. The requirement for subcellular targeting of viral proteins will be determined by expressing wild-type and mutant proteins individually in infected protoplasts and plant cells using a plus-strand RNA viral vector. Objective 2 will involved developing a genetic analysis system amenable to studying SYNV replication, and to identify the host components that are required for infection. Three viral proteins required for transcription and replication of the minus-sense genomic RNA will be expressed in yeast. The SYNV derivatives containing a selectable marker gene to complement an essential mutation will be evaluated for infectivity. Successful completion of these objectives will provide enormous potential for molecular genetic analysis of both virus and host components and will have considerable applicability to other minus-strand viruses.